Antenna movements as a function of odorants’ biological value in honeybees (Apis mellifera L.)

In honeybees, the antennae are highly mobile sensory organs that express scanning movements in various behavioral contexts and toward many stimuli, especially odorants. The rules underlying these movements are still unclear. Using a motion-capture system, we analyzed bees’ antennal responses to a panel of pheromonal and other biologically relevant odorants. We observed clear differences in bees’ antennal responses, with opposite movements to stimuli related to opposite contexts: slow backward movements were expressed in response to alarm pheromones, while fast forward movements were elicited by food related cues as well as brood and queen related pheromones. These responses are reproducible, as a similar pattern of odor-specific responses was observed in bees from different colonies, on different years. We then tested whether odorants’ attractiveness for bees, measured using an original olfactory orientation setup, may predict antenna movements. This simple measure of odorants’ valence did however not correlate with either antennal position or velocity measures, showing that more complex rules than simple hedonics underlie bees’ antennal responses to odorants. Lastly, we show that newly-emerged bees express only limited antennal responses compared to older bees, suggesting that a significant part of the observed responses are acquired during bees’ behavioral development.

www.nature.com/scientificreports/ Because of the high correlation between the two years' datasets, they were pooled for further analysis. The resulting united dataset retained strong stimulus effects for both angle (Fig. 3A, RM-ANOVA; stimulus effect: F 15, 360 = 2.87, p < 0.001) and velocity (Fig. 3B, RM-ANOVA; stimulus effect: F 15, 360 = 2.20, p < 0.01). We then analyzed the link existing between changes in angular position and velocity in response to the odorants. We found a significant negative correlation between Δθ and ΔVθ ( Fig. 3C; Pearson correlation, R 2 = 0.40, p < 0.01), suggesting that generally, odorants that brought the antennae forward also increased their velocity and conversely, odorants that brought the antennae backward tended to slow them down.
Attractiveness of the odorants. We next asked whether honey bees' antenna movements relate to their hedonic evaluation of the odorants. To address this question, we developed a high-throughput assay for measuring each odorants' attractiveness, and measured the movements of about 800 bees in the dark, each confronted to one of our stimuli. The setup consisted in individual 45 cm glass tubes, with a small box containing a bee at one end, and a small box containing a filter paper with a particular odorant (or control) at the other (Fig. 4A). Three equally spaced automatic infrared light portals were positioned along the tube (Trikinetics, Walham, MA, USA, see methods). The movements of the bee through the three portals were monitored for 10 min and an orientation index (OI) was calculated from the number of passages through the monitors:  Fig. 2A,B). Octanoic acid induced a forward motion of the antennae with an acceleration, whereas 2-heptanone induced a backward motion of the antennae with a deceleration. The changes in angular position (Δθ) or angular velocity (ΔVθ) were calculated as the difference between these values during odor presentation (5 s) and before (15 s www.nature.com/scientificreports/ M1 being the monitor close to the bee's initial position and M3 the monitor closest to the odorant box. The control group was presented with an odorless box. To measure the relative attractiveness and repulsion of each odorant, the average orientation index of the blank control group was subtracted from that of each odorant. This way, a positive attractiveness index (AI) indicated an attracting odor whereas a negative index indicated a repellent odor (Fig. 4A). In agreement with the widely differing sources and inferred biological values of our odorants (see Table 1), we observed a significant heterogeneity in the AIs of the different odorants ( Fig. 4B: ANOVA; stimulus effect: F 14, 600 = 2.71, p < 0.001). Only two odors produced by the queen and the brood showed a positive attractiveness index: QMP and ethyl oleate. The aggregation pheromone compound geraniol produced only a slight attraction. The rest of the odorants were neutral or acted repulsively.
We then plotted the two variables of antennal movements, Δθ and ΔVθ, as a function of attractiveness values (Fig. 4C,D). Weak trends seemed to emerge, with odorants' attractiveness suggesting forward and quicker antenna movements. However, possibly due to the limited number of odorants, we found no significant correlation between AI and Δθ (Fig. 4C, Pearson correlation, R 2 = 0.17, p = 0.124) or between AI and ΔVθ (Fig. 4D, R 2 = 0.17, p = 0.126). Note that this result was the same when only the first year's antenna movement dataset (Δθ vs AI: R 2 = 0.15, p = 0.14; Δθ vs AI: R 2 = 0.19, p = 0.10) or only the second year's antenna dataset (Δθ vs AI: R 2 = 0.16, p = 0.14; Δθ vs AI: R 2 = 0.07, p = 0.34) were used. We conclude that honeybee's antennal movements to odorants cannot be directly predicted by a simple measure of odorant attractiveness.
Concentration influence on the antennal responses. We next wondered how odorant quantity affected antennal responses and attractiveness measures. Because the same volume of pure odorant was used for almost all stimuli, the absolute concentration in the air flow depended on its vapor pressure, which was different among our odorants (Suppl. Fig. S2A). Indeed, we found that antennal position (Δθ) was significantly correlated to odorants' vapor pressure, i.e. more volatile odorants (with a higher vapor pressure and accordingly presented at higher concentration) tended to induce backward antenna movements (Suppl. Fig. S2B, Δθ vs VP log , Pearson correlation, R 2 = 0.63, p < 0.01). By contrast, neither antenna velocity (Suppl. Fig. S2C, ΔVθ vs VP log , R 2 = 0.20, NS) nor attractiveness correlated with odorants' vapor pressure (Suppl. Fig. S2D, AI vs VP log , R 2 = 0.04, NS). Based on these observations, we next tested how odorant concentration may affect antenna movements. We selected three odorants that induced remarkable changes in antennal movements in previous experiments: the defense compound, 2-heptanone, the aggregation compound geraniol, and the royal jelly odorant, octanoic acid ( Fig. 5). They were presented to the bees at eight different concentrations ranging from 10 -7 to 10 0 , in increasing order.
Antennal responses in newly emerged bees. To further evaluate the possibility that antennal responses to odorants are acquired in the course of honeybees' adult life, we analyzed them in newly emerged bees. Bees that had emerged from their comb cell in the last 24 h were stimulated, together with an air control, with a selection of five olfactory stimuli which had induced contrasted responses in older bees in the previous experiments: two alarm/defense pheromones (2-heptanone, isopentyl acetate), one brood pheromone (methyl linoleate), the queen pheromone (QMP), and the royal jelly odor (octanoic acid). The changes in angular position (Δθ) and velocity (ΔVθ) recorded during odorant stimulations are presented in Fig. 6. Newly emerged bees did not orientate the antennae in response to the different odorants, as no contrast among stimuli appeared for Δθ (stimulus effect: F 5, 120 = 0.81, NS). However, newly emerged bees increased antenna movements when odorants were presented, with a significant heterogeneity observed among stimuli for ΔVθ (stimulus effect: F 5, 120 = 7.54, p < 0.001). Almost all odorants tested induced an increase in velocity (2-heptanone, isopentyl acetate, octanoic acid, QMP; Dunnett tests, p < 0.05; exception methyl linoleate, NS).

Discussion
In this study, we observed odor-specific antennal movements to a range of pheromones and general odorants with different biological values for honey bees. Bees' responses recorded from two colonies, on two different years, were correlated, showing that antennal responses to odorants are reproducible. Generally, odorant-induced changes in antennal position and velocity were correlated, so that the more the antennae were brought to the front, the more quickly they moved. Building an original olfactory orientation setup, we observed clear differences in tested odorants' attractiveness to the bees. However, odorants' attractiveness, as measured in our setup, did not correlate significantly with either antennal position or velocity measures. Lastly, we show that the antennal responses of newly-emerged bees are limited compared to older bees. While the tested odorants induced an acceleration of antenna movements like in older bees, they did not produce any change in antenna position. We included in our experiments a few odorants that had been tested in previous recordings of antenna movements 50,51 . In Erber et al. (1993) 50 , bees' antenna movements were recorded thanks to two photodiodes, each one located in front of one of the bees' antennae (at an approximately 45° angle in our coordinate system, see Fig. 1B therein). An 'antennal response' in this study corresponded to an increased frequency of antennal passages on the diodes during odor presentations. Bees 'responded' to geraniol and citral (aggregation pheromone components) and to octanoic acid (therein termed caprylic acid, a major royal jelly volatile) but not to isopentyl acetate (alarm pheromone). In this previous work, it was however not possible to know if bees kept their antennae more to the front and/or increased their antennal scanning velocity during a response. The use of a camera-based system in our study allowed disentangling these effects. Fitting with Erber et al. (1993) 50 , octanoic acid produced in our recordings accelerated movements to the front. Citral and geraniol slightly increased antennal speed (about 1°/s), even if they produced contrasted changes in antenna position (Fig. 3A,B). Lastly, although isopentyl acetate induced an increase in antennal speed (Fig. 3B), it brought the antennae strongly to the back of the bees' head ( Fig. 3A), explaining the lack of response in Erber et al. (1993) 50 . We acknowledge that, like formerly described systems 51,53,54 , our recordings focused on the measure of antennal movements in the transverse plane of the honey bee head, thus in two dimensions. This persisting choice in the studies of antennal movements is due to the observation that most of the bees' antennal movements take place in this plane. We are therefore confident that the changes in antennal movement observed in the present study represent a prominent and relevant part of the bees' antennal behavior during odor scanning. Developing a three-dimensional recording strategy by using two or more motion capture systems placed around the bee's head and by temporally synchronizing their dataflows is possible and some recent efforts were made in this direction 55 .
Thanks to the use of a wide odorant panel, we showed that odorants induce diverse antennal responses, with both forward and backward movements, and both increased and decreased velocities. Interestingly, position and velocity changes in response to odorants were correlated (Fig. 3C) and bees' antennal responses could roughly be separated in two groups: fast-forward movements and slow-backward movements. It is possible that the observed correlation between position and velocity measures is purely mechanical, and related to the structure of bees' antennal muscles. When taking into account the known biological value of these odorants for bees, interesting general tendencies emerged. While the slow-backward movements were mostly expressed in response to alarm/ defense pheromones (see red dots in Fig. 3C), especially to 2-heptanone, fast-forward movements were rather elicited by food-related odors (octanoic acid, the royal jelly odor, blue, and octanal, grey), pheromone components linked to the signaling of valuable resources (geraniol, an aggregation pheromone component, green), as well as social signals like brood and queen pheromones (light and dark violet, Fig. 3C). There were some exceptions to these rules, like the recorded backward antennal response to citral (an aggregation pheromone component) or β-ocimene (a volatile brood pheromone compound). Similarly, some odorants with a strong inferred biological value, like the fecal compound 3-methyl indole (scatol), did not induce strong antennal responses. This being said, fast forward movements to food-related odors appear consistent with previous studies showing that sucrose, or odorants previously associated with sucrose, induce forward antenna movements 50 www.nature.com/scientificreports/ other hand, slow/backward antenna movements to alarm/defense compounds seems coherent with a defensive context, where responding to appetitive stimuli is of secondary importance, and protecting important sensory organs like the antennae may be more appropriate. In addition, the strong backward response to 2-heptanone somehow fits with its use by bees as a deterrent to mark depleted flowers 59,60 . Clearly odorant quantity had an effect on antennal responses, because antennal position (but not velocity) varied as a function of odorant's vapor pressure. When testing three odorants at eight different concentrations we found that both position and velocity varied with odorant concentration. Antennal responses generally started at 10 -3 concentrations, which corresponds to concentrations at which clear odor-induced neural activity is observed in the antennal lobe in optical imaging experiments [61][62][63] . Interestingly, antennal responses did not simply increase monotonously with concentration. Remarkably, bees' antennal responses to geraniol were stronger at medium than at high concentration. This possibly relates to the known dose-dependent effects of pheromones on behaviour [64][65][66][67] and the fact that in natural situations, pheromones are used within a definite concentration range. It is thus possible that given concentrations best evoke odorants' pheromonal value for bees and therefore trigger stronger antennal responses than higher concentrations. In any case, odorant concentration affected the amplitude of the response, but not its direction. We did not observe any opposite responses (forward/backward or slower/faster) for the same odorant at different concentrations.
Opposite influences of pheromones with differing biological values on antenna movements as observed in our experiments are remarkable in the context of current debates on pheromones' behavioral side-effects. It has been increasingly suggested that pheromones, in insects but also in mammals, can act as modulators of a variety of behavioral responses which are not the primary -known-targets of their action 68,69 . In honey bees, some alarm pheromone components decrease bees' responsiveness to an appetitive reward like sucrose 69 and negatively impact appetitive learning performances 67,[70][71][72] . Conversely, an aggregation pheromone component, typically associated with appetitive behavior, has been shown to decrease responsiveness to a noxious stimulus like an electric shock 73 and to improve appetitive learning 72 . The model extracted from these findings posits that pheromones-or odorants with a strongly innately attached value-modulate the bees' internal state relative to two main modules, an appetitive module and an aversive module ('defensive and appetitive scores' 73 ). It classifies pheromones in clear categories along a common hedonic dimension, with alarm pheromones bearing a negative ('defensive') value and aggregation pheromones (or floral odorants) bearing a positive ('appetitive') value. Accordingly, alarm pheromones reduce the appetitive score and aggregation pheromones reduce the defensive score. The contrasted antennal responses we observed may represent behavioral clues for the existence of such opposite odorant values. We attempted to capture such a hedonic dimension in our odorants by measuring their attractiveness for bees in an olfactory orientation setup (Fig. 4). However, correlation coefficients between attractiveness indices and antenna movement variables were not significant (p = 0.12 for both angle and velocity), even if the figures suggest a possible trend, with attractive odorants corresponding more to fast and forward antenna movements. Possibly, including more odorants in future studies could provide more statistical power for demonstrating a link between both variables. Note however, that the real-life situation is more complex than the simple hedonic model presented above, since not all pheromonal components of a given type have the same effect on behavioral responses. For instance, 2-heptanone, but not isopentyl acetate affects responsiveness to sucrose 69 , whereas isopentyl acetate, but not 2-heptanone, affects responsiveness to an electric shock 73 . Likewise, in our data, the brood pheromone β-ocimene brought the antennae to the back, while the other brood pheromones (ethyl oleate and methyl linoleate) brought them to the front (Fig. 3A). Thus, bees' evaluation of odorants may be best described on more than one simple dimension, as each conveys a different message, usually presented in a particular context.
We evaluated the reproducibility of antennal responses to odorants by measuring them on bees from different hives on different years. We found a clear correlation between the two years' datasets, both in terms of antenna position and velocity. This result may indicate that some of these responses (in particular to pheromones) are innate. Our observations suggest however a strong importance of bees' experience. While some odorants induced very similar responses on both years, others induced remarkably different behaviors. Octanoic acid, for instance, produced strong forward and accelerated movements on the first year, but only weak responses on the second year. This points to an effect of experience, which we demonstrated in a previous study where odorants associated with food suddenly induced fast forward antennal movements 52 . In fact, finding the same pattern of odor-specific responses on the two years is not a proof per se that antennal responses are innate, because the observed patterns could simply be the result of our odorants being associated with similar contexts and consequences during the lives of these two groups of bees. To understand the ontogeny of these odor-induced responses, we recorded antenna movements in newly emerged bees. We found rather limited antennal responses show the replication of the experiment on the following year (N = 25). Color code: air control (white), alarm pheromones (red), aggregation pheromones (green), brood pheromones (light purple), queen pheromone (dark purple), floral odors (grey), repulsive odor (orange), fecal odor (brown), and the royal jelly component (blue). Asterisks in the square next to the graph indicate a significant heterogeneity in antennal movements between odorants (RM-ANOVA, ***: p < 0.001). Asterisks on the histograms indicate significant differences in Dunnett post-hoc tests comparing each value to the air control (•: p < 0.1; * = p < 0.05). (E,F) Regressions comparing the results of the two experimental years in terms of (E) antennal angular position (θ) and (F) angular velocity (ΔVθ). Asterisks in the square next to the graph indicate significance in a Pearson correlation test (***: p < 0.001). www.nature.com/scientificreports/ at this age, especially regarding angular position changes. This suggests that odor-specific antenna movements are acquired by the bees in the course of their adult life. This could be due to an incomplete maturation of their antennal motor abilities when emerging, but could also relate to their behavioral development in the context of bees' age polyethism. Interestingly, newly emerged bees did not show the specific slow-backward movements to alarm pheromones found in older bees. This is consistent with the fact that bees' aggressiveness and response to alarm pheromones increases with age 74 , paralleling the ontogeny of defensive behavior 75,76 . This behavioral development is accompanied by changes in biogenic amine and hormone titers [77][78][79] . For instance, the levels of juvenile hormone and octopamine increase with age [80][81][82] . A role of biogenic amines in particular is supported by the observation that octopamine and serotonine have opposite effects on antennal movements, increasing and decreasing them respectively 58 . Thus, part of the age effect we found could be related to differences in levels of these biogenic amines. Our study thus gives some insights into the effect of age on odor-evoked antennal movements, but another aspect that would be extremely interesting to study is the effect of the bees' tasks. Since younger individuals perform tasks within the colony while older individuals engage in tasks outside of the hive, we may consider that our experiments described the two ends of the task spectrum of a normal colony. Future experiments should however disentangle the respective effects of age and task on antennal responses to taskrelated odors and pheromones. Different odorants induce different antenna movements according to their biological value for bees. This, together with the observation that antennal movements are modified by associative conditioning 52   www.nature.com/scientificreports/ by different muscle groups moving the antenna scape (4 muscles) and the flagellum (2 muscles). Motor neurons controlling this muscular system originate in the AMMC (antenna mechanosensory and motor center) [83][84][85][86] . Response to tactile stimuli is thought to use a short route as mechanosensory neurons project directly to the AMMC 83 . Antennal reaction to olfactory stimuli, by contrast, should take a longer route. Odorants are detected by olfactory sensory neurons in the antenna, which relay odor information to a primary olfactory centre, the antennal lobe (AL), composed of glomeruli, which each receives input from OSNs (Olfactory Sensory Neurons) expressing the same olfactory receptor type. The AL processes olfactory information and second-order (projection) neurons (PN) transmits it to higher-order brain centers, the mushroom bodies (MB) and the lateral horn (LH). Honey bee pheromone compounds (alarm, aggregation, brood, queen, etc.) all trigger combinatorial activity from many glomeruli in the worker antennal lobe 87,88 . This suggests that their biological value is extracted within higher-order centers 89 . Indeed, projections from the AL to the honey bee LH were shown to contain combinatorial information allowing to differentiate the different pheromone types 90 . The LH is considered as a www.nature.com/scientificreports/ premotor center mediating fast and innate reactions to biologically relevant stimuli, and may be responsible for innate antennal movements to odorants. Direct connections between the LH and the AMMC are not described yet in honey bees, but they are known in fruit flies 91,92 . Changes in odor-induced antennal responses through experience (like after associative conditioning) would involve the MB, the learning and memory center of the insect brain. In the MB, the Kenyon cells (KC) are highly odor-specific and are activated by the combinatorial input from many different PNs 93 . Information from KCs is read out by MB-output neurons which project to different parts of the protocerebrum, including the LH. MB-output neurons are plastic and their odor-induced responses are modified by experience [94][95][96][97] . Some of them, like the PE1 neuron, project to the LH 95,98 and may be responsible for an experience-driven modulation of odor-induced antennal movements through this structure. To our knowledge, no direct connections of MB output neurons to the AMMC have been described 98 , but indirect pathways other than through the LH are possible.  www.nature.com/scientificreports/ To conclude, honey bees display a range of different antennal responses to odorants, which vary as a function of odorants' biological value. These responses are reproducible, suggesting that they are in part innate, but they are also shaped by the bees' experience and develop in the course of their lives. A necessity of our approach, but a clear limitation nonetheless, is that bees' responses were recorded in an experimental context quite different from natural situations. In other insects, social context in particular has been shown to modulate behavioral responses to olfactory stimuli 99,100 . Bees' antennal response to alarm pheromones, for instance, may be quite different when they are guarding at the hive entrance. A next step should thus be to analyze antennal movements in more natural, hive, situations.

STAR★methods
The material used in this article is listed in Table 2.
Contact for reagent and resource sharing. Further information and requests for resources, data and reagents should be directed to and will be fulfilled by the Lead Contact, Jean-Christophe Sandoz (sandoz@egce. cnrs-gif.fr) and Hanna Cholé (hanna.chole@gmail.com).

Experimental model and subject details.
The experiments were carried out on adult honey bee workers (Apis mellifera) captured at the hive entrance on the CNRS campus in Gif-sur-Yvette (France) in April-May 2015 and 2016. The newly emerged bees used in the last experiment (Fig. 6) were caught on the morning of the experiment when emerging from a brood comb placed the day before in an incubator (at 35 °C). Bees were chilled on ice until they stopped moving in order to harness them individually in plastic holders, leaving their antennae and mouthparts free. They were then fed with 5 µl sucrose solution (50% w/w), 4 h before the beginning of the experiments. After feeding, a drop of color was applied on bees' antennae tips, using water-based paint (Posca PC-5 M, Mitsubishi Pencil Co.). Bees were marked on the upper surface of the last two flagellomeres, to allow the motion capture system to record their coordinates (see below), without impairing olfactory perception see 52 . Once mounted, fed and marked, individuals were placed in a moist, dark polystyrene box, until the start of the experiments.

Method details
Antenna monitoring apparatus. The recording apparatus was composed of a camera positioned above the bee holder (Fig. 1A). The camera included an integrated processing card allowing adaptive detection (using a motion prediction algorithm) of the two color dots, up to a rate of 120 Hz (BIPcam, Brain Vision Systems). The camera managed to follow and record the coordinates of the color dots on the antenna tips, in real time at a rate of 90 Hz. In order to optimize the detection of the color dots, the apparatus was placed in a room with low light conditions (controlled and kept constant). A cold light illumination ring was placed around the lens of the camera, diffusing homogeneous white light on the bee's head (Leica CLS 150XE, Leica, Jena, Germany). The intensity of the light source was tuned precisely and kept constant for the duration of the experiments.
The olfactory stimulation apparatus provided a constant air flow of 52.5 mL/s. This flow, composed of a principal air flow of 50 mL/s and a secondary flow of 2.5 mL/s, was directed to the bee by a glass tube (0.5 cm diameter), at a distance of 2 cm. The secondary air flow could be directed to one of two sub-circuits (one containing an odorant source, and another without any odorant) before being reinjected into the main airflow. Most of the time, air flowed through the odourless sub-circuit. Olfactory stimulation was applied manually inducing a switch of the secondary flow to the odorant sub-circuit for 5 s. The odorant sub-circuit included a Pasteur pipette containing the odor source (see below). The other sub-circuit included an identical Pasteur pipette without odorant. An air extractor, placed behind the bee prevented odorant accumulation.
Antenna movement analysis. Before the recording period, each bee was left to acclimatize for 20 s to the apparatus. Each recording lasted 60 s. The monitoring apparatus recorded at each time point (90 times per second) the location of the two antenna tips of each bee on the camera sensor (pixel coordinates). First, all the recordings from all bees were recalculated in the same coordinate system (x,y), with the socket of the right www.nature.com/scientificreports/ antenna as the origin (coordinate 0,0) and the socket of the left antenna as the unit reference on the x-axis (coordinate 1,0). Each recording thus resulted in a series of (x,y) coordinates for each antenna at each time-step (1/90 s). This allowed a comparison between the antennal movements of different bees. Previous studies 51,52 showed that bees' antennal movements are best described using circular coordinates (r, θ), as each antenna moves around its socket (Fig. 1B). Thus, each antenna's movements were described in their own coordinate system, with the antenna socket (base) as the origin (0,0).
• Angular position (θ): it was defined as the angle between a line connecting the antenna tips to their base (r) and an anteroposterior line passing through the corresponding antenna base. This variable indicates if the antenna is positioned to the front (0°), to the side (90°) or backward (180°). Note that the measured angle is symmetrical for the left or the right antenna so that 90° is on the left for the left antenna and on the right for the right antenna. • Distance to antenna base (r): it was defined as the distance between the antenna base and the antenna tip.
This variable thus measures whether the antenna is in a stretched or retracted position. • Angular velocity (Vθ): it was calculated as the angle θ traveled by each antenna during a frame (1/90 s). It is expressed in degrees per second.
As explained in the results, θ and Vθ proved to be the most pertinent for measuring changes induced by conditioning and are thus presented in the figures. The r data are presented in Supplemental Material (Suppl. Fig S1). The response to each odorant was calculated as the change between the antennal movements before and during the odorant stimulation. Thus, Δθ (resp. ΔVθ) was calculated as the average of θ (resp. ΔVθ) during the stimulus minus the average of θ (resp. ΔVθ) before the stimulus (Fig. 1C,D).
In most experiments, the two antennae of the bees were marked and recorded, and angle and velocity data were averaged between antennae before any analysis. In the concentration experiment (Fig. 6), only one antenna per bee (balanced between right and left) was tracked allowing faster data analysis. Since previous studies observed different behavioral and/or neurophysiological effects depending on the side of olfactory stimulation (through the right or left antenna [116][117][118] , we statistically assessed whether antenna movements differed between sides. We found no effect of antenna side, neither for Δθ (RM-ANOVA, F 1,41 < 3.29, p > 0.077) nor for ΔVθ (RM-ANOVA, F 1,41 < 0.31, p > 0.579).

Odorants for antenna movement analysis. Up to fifteen odorants with different biological values for
bees were tested in the experiments. They are detailed in Table 1. For most stimuli, 5 μL of the odor solution were placed on a filter paper strip inserted in a Pasteur pipette. The odorants were used pure except for 3-methyl indole, which was a powder diluted in water at a concentration of 0.48 mg L −1119 . The queen pheromone QMP was presented as a commercial stick (BeeBoost ®, Pherotech, Delta, Canada), directly inserted into a Pasteur pipette. Except for this last stimulus, all odorants were obtained from Sigma-Aldrich (Deisenhofen, Germany). As control stimulus, a pipette containing a clean piece of filter paper was used. The order of odorant presentations was randomized between bees. In one experiment, three odorants (geraniol, octanoic acid and 2-heptanone) were tested at eight concentrations ranging from 10 -7 to 10 0 . They were prepared in mineral oil and were presented in increasing concentration order to avoid adaptation (i.e. the 10 -7 concentrations of the three odorants were presented to the bee before moving to the 10 -6 concentrations, etc.). The control stimuli were a pipette containing a piece of filter paper soaked with 5 μL mineral oil and a pipette with a clean piece of filter paper. Both were presented before and after the odorant concentrations. The interval between odor presentations was ~ 60 s.

Odorant attractiveness measure.
To determine the attractiveness of each odorant for bees, we developed a high-throughput assay allowing to simultaneously monitor the movements of 16 bees, each confronted to a different stimulus (Fig. 4A). Each of the 16 lines of the setup was 45 cm long, made of 3 connected glass tubes (12 mm internal diameter), with two plastic (3D printed) boxes placed at each end, one containing the bee and the other containing a filter paper with 5 µL of pure odorant. The bee box contained a sliding door allowing to control when each bee was allowed to enter the glass tube. Three automatic, equally spaced infra-red portals (Trikinetics, Walham, MA, USA), allowed counting the passages of each bee at three locations along the tube. The apparatus was placed in the dark to ensure that bees' movements were based on olfaction, and under an air extractor to prevent any accumulation and contamination of odorants. Sixteen bees were tested simultaneously, each with one of the 15 odorants or with an odorless box (control). Each bee was allowed to enter its tube by opening the door of its box. Her movements through the three portals were then monitored for 10 min. Bee boxes and the glassware were thoroughly washed between trials, while odorant boxes were kept separate in plastic bags and always contained the same odorant throughout the experiment.
In the end, an orientation index was calculated from the number of passages through the portals using the following formula: (M3-M1)/(M3 + M1), were M1 is the monitor close to the initial position of the bee, the farthest from the odorant box, and M3 is the monitor the closest to the odorant box. Each bee was used only once in the apparatus, with only one odor. The raw position index of the control group (odorless box) was subtracted from that of each odorant to obtain this odorant's attractiveness index (AI). A positive AI (Fig. 4) thus indicated an attractive odor whereas a negative AI indicated a repellent odor.
Statistical analysis. Before analysis, the normal distribution of antennal and attractiveness data were confirmed with Shapiro-Wilks tests. To compare antennal responses (changes in angular position Δθ and velocity ΔVθ) among odorants, repeated measure analyses of variance (RM-ANOVA) were used, with stimulus (odor-Scientific Reports | (2022) 12:11674 | https://doi.org/10.1038/s41598-022-14354-z www.nature.com/scientificreports/ ants including air control) as within group factor. When significant, Dunnett post-hoct tests allowed to compare each odorant's value to the control. In the odorant concentration experiment, a RM-ANOVA with stimulus and concentration as within-group factors was used. If significant, it was followed by individual RM-ANOVAs for each odorant, with only concentration as a within-group factor. When significant, individual concentrations were compared to a common control (average of the four air/mineral oil stimulations), using Dunnett tests.
To make sure there were no difference between the groups marked in the right or left antenna, a RM-ANOVA with antenna side as categorical factor was used, on all the odors and concentrations, as well as on the controls separately. Concerning the odorant attraction assay, differences in attractiveness index were compared among stimuli using an ANOVA (note that different bees were tested with each odorant). Pearson correlation tests were used to evaluate the relationships between the attractiveness index, odorants' vapor pressure or concentration and the antennal movement variables Δθ and ΔVθ. All statistical analyses were performed with Statistica® 7.0 software (StatSoft, Inc. 2004).

Data availability
The data are available on request from the authors.